Synthesis, characterization and antifungal studies of metalloquinolone [Cd₂(nal)₂(phen)₂(Cl)₂]

Abstract

A novel dinuclear, distorted octahedral complex of nalidixic acid (nal) with Cd(II) metal ion with the formula [Cd₂(Nal)₂(Phen)₂(Cl)₂] has been synthesized in the presence of N-containing heterocyclic ligand, 1,10-phenanthroline (phen). The synthesized metal complex was characterized using CHN analysis, Fourier transformed infra-red, thermo gravimetric analysis, differential scanning chalorimetry, nuclear magnetic resonance, ultra violet-visible and single-crystal X-ray diffraction. The newly synthesized complex shows more pronounced antifungal activity compared with the parent quinolone against four fungi, namely Pythium aphanidermatum, Sclerotinia rolfsii, Rhizoctonia solani and Rhizoctonia bataticola.

• Metal complex
• Metalloquinolone
• Single crystal

1. Introduction

Quinolones exhibit excellent antibacterial results to both gram-negative and gram-positive bacteria [1]. In clinical practice, the introduction of nal (Figure 1), first member of quinolone group, causes ease for the treatment of various infections, such as urinary tract infections, soft tissue infections, respiratory infections, bone-joint infections, typhoid fever, sexually transmitted diseases, prostatitis, community-acquired pneumonia, acute bronchitis and sinusitis [2,3]. Nal selectively inhibits the DNA replication in microbes due to the presence of 4-oxo and 3-carboxyl groups. These two groups interact with guanine of susceptible microbial DNA in the gyrase–DNA complex. Then the resulting quinolone–enzyme–DNA complex stops the progress of the normal replication fork and finally causes microbial cell death [4,5].

Fig. 1. Chemical structure of nal.

Recently, a new approach for the designing of antimicrobial agent has been explored using some metal complexes of quinolone that display a novel mode of action due to the synergetic effect of metal [1]. The close proximity of carboxylate and pyridone oxygen on the quinolone molecule would account for its good chelating properties. Several quinolone metal chelates are known to possess antibacterial, antifungicidal, antiviral and anticancer activity and that is why the study of interaction between quinolone and metals becomes an active area of research in bioinorganic chemistry [4,6,7].

It has been observed that metal complexes with appropriate ligands are biologically more significant and specific than the metal ions and the ligand itself [8–10]. A real breakthrough in the isolation of quinolone-mixed ligand complexes was achieved by using hydrothermal reactions introduced by the group of Xiao-Zeng You [11–13]. In the literature, various metal complexes having single-crystal structures with different quinolones have been isolated [14–39].

To enhance the knowledge of the behavior of nal as a complexing agent in a biological point of view, we have synthesized a novel mixed ligand complex of Cd(II) in the presence of another biologically active N-containing heterocyclic compound phen. Although cadmium is a heavy, toxic metal, it has been checked for the antibacterial ability of several cadmium complexes of different quinolones as the complexes of soft-acid metal ions are more effective antimicrobial agents than others [40].

2. Experimental

2.1 Materials

Nal was purchased from Sigma-Aldrich. All chemicals used are of analytical grade.

2.2 Synthesis of complex

Sodium nalidixate was synthesized prior to the experiment to increase solubility of nal. The Cd(II) complex has been synthesized by mixing 5 mL of 0.5 mM aqueous solution of phen and 5 mL of 0.5 mM aqueous solution of . The mixture was stirred on a magnetic stirrer for 10 min at room temperature. Then the resulting mixture was added to 5 mL of the aqueous solution containing 0.5 mM of nalidixic acid and 0.5 mM of NaOH after adjusting its pH 7.0. Then the overall mixture was heated in a hydrothermal vessel at 100°C for 24 h followed by the slow cooling of the vessel for 5 days. Heating and cooling have been carried out in an oven of programmed temperature. Finally, a yellowish white crystal was obtained.

Calc. for C₅₃H₅₆Cd₂Cl₂N₈O₆: C, 51.60; H, 3.668; N, 9.937%. Found: C, 51.45; H, 3.60; N, 10.00%.

2.3 Physical measurements

Fourier transformed infra-red (FT-IR) spectra were recorded on a spectrometer Perkin Elmer Spectrum BX II in the range of 400–4000 cm⁻¹ by preparing sample pellets with KBr. Electronic spectra were recorded in solid state on a instrument Shimadzu UV-3101PC spectrometer. C, H and N elemental analysis was performed on an instrument named Elementer vario ELIII. Thermo gravimetric analysis (TGA) measurements were carried out in an oxygen atmosphere from ambient temperature to 900°C using Perkin Elmer Diamond. Nuclear magnetic resonance (¹H NMR) spectra were recorded with the help of a Jeol-FT-NMR spectrometer. Single-crystal X-ray diffraction (XRD) was recorded with a diffractometer, Bruker D8 using Cu Kα radiation.

2.4 Microbiological studies

Bio-efficacies of the synthesized complex were checked in vitro. The in vitro antifungal activities of the ligand and the complex have been evaluated against four pathogenic fungi, Pythium aphanidermatum (PA), Sclerotinia rolfsii (SR), Rhizoctonia solani (RS) and Rhizoctonia bataticola (RB) by the agar plate technique. The compounds are directly mixed with the medium in 0, 12.5, 25, 50 and 100 ppm (in Dimethyl sulfoxide) concentrations. Controls were also run and three replicates were used in each case. The linear growth of the fungus was obtained by measuring the diameter of the fungal colony after four days and the percentage inhibition was calculated by the following equation: where C and T are the diameters of the fungal colony in the control and the test plates, respectively [41].

3. Results and discussion

3.1 Infra-red spectroscopy

FT-IR band assignments of the newly synthesized complex were compared with those of standard ligand, nal to determine the coordination mode of the ligand. FT-IR spectrum of nal shows carboxylic stretch at 1711 cm⁻¹ and pyridone stretch at 1614 cm⁻¹. The position and intensity of the peaks were found to be changed upon chelation. In the spectrum of the synthesized complex, carboxylate stretch has been replaced by two strong characteristic bands which are observed at 1582 and 1426 cm⁻¹ assigned as asymmetric and symmetric stretching vibrations, respectively, where was shifted from 1614 to 1627 cm⁻¹ due to the bonding with metal ions (S1). The difference is the important criteria for the determination of coordination mode of the ligand [42]. In the present case, Δ ν value for the synthesized complex is 156 cm⁻¹, indicating the bridging mode of coordination of the carboxyl group of nal. The overall changes in the FT-IR spectrum concluded that nal behaves as a bridging ligand and binds to the metal ions through pyridone oxygen and one of the carboxylate oxygens which make bridge with another Cd(II) ion.

3.2 ¹H NMR spectroscopy

Further evidence for the coordinating mode of the ligand was obtained from ¹H NMR spectra. The ¹H NMR spectra of ligand and complex were recorded in deuterated dimethyl sulfoxide at 400 MHz. Full assignment of every signal in the ¹H NMR spectrum of the complex was achieved by comparing the spectrum of the ligand. ¹H NMR spectra (S2) of ligand exhibit a broad peak at δ14.59–14.88 ppm due to carboxylic proton. This signal of the ligand disappears in the complex. The absence of this signal in the complex suggests that this has been lost due to complexation. Comparison of ¹H NMR spectra of the complex with that of nal shows a significant perturbation of the chemical shifts of the aromatic protons indicating that they are adjacent to the coordinating sites of the ligand. In the case of metal complex, four new peaks

are generated in the aromatic region due to the presence of phen. The overall changes of the ¹H NMR spectra of the complex are indicative of coordination of nal to the metal ion via pyridone oxygen and one carboxylate oxygen. From ¹H NMR spectra, it is also evident that there is no change in the complex structure in deuterated dimetyl sulfoxide.

3.3 Electronic spectra

Electronic spectra of the Cd(II) metal complex and its ligand were recorded in the wave length region of 200–900 nm in solid state. In the case of the parent ligand, two bands have been observed at 285 and 335 nm assigned for π−π* and n−π* transition, respectively. These two bands are due to the presence of aromatic ring having pyridone oxygen and carboxylate oxygen. The pattern of the spectra of the synthesized complex was similar to that of nal which reveals that the structure of the ligand remains the same during complexation; differences due to phen are not easily distinguished [43–46]. But these two spectra only differ in intensity. Both the bands in the spectrum of the complex are shifted hypochromically compared with the free ligand indicating the participation of the pyridone oxygen and carboxylate oxygen during complexation which is in accordance with FT-IR results also. An additional broad band was observed (S3) in the case of synthesized complex, centered at 450 nm due to metal to ligand charge transfer spectra.

3.4 Thermal analysis

Thermal analysis of metal complex was investigated from room temperature to 900°C under oxygen atmosphere with a controlled heating rate of 10°C min⁻¹. The temperature ranges, percentage weight loss and eliminated moiety in each decomposition are listed in Table 1. The synthesized complex was decomposed in two steps where nal was decomposed in a single step (Figure 2). During the first stage of decomposition of the complex, 46.93% of experimental weight loss (calculated weight loss is 46.75%) was observed in the temperature range 257–457°C with the elimination of the moiety C₂₆H₂₆CdClN₄O₃. In this stage of decomposition of the synthesized complex, two bridging Cd–O bonds breaking were observed. In the case of nal, it is 92.44% weight loss in the temperature range 203–357°C. In the second stage of decomposition, 35.29% (calculated weight loss is 36.37%) of weight loss was observed in the temperature range of 457–899°C with the elimination of one molecule of phen, oxygen and chlorine and finally CdO was left as the residue. It has been also found that the melting point of the synthesized complex (288.9°C) is more than that of parent quinolone (232°C). From the above thermal study, it is obvious that the synthesized complex is more thermally stable than its parent ligand nal.

Table 1  Thermo gravimetric data of (a) nal and (b) [Cd₂(nal)₂(phen)₂Cl₂].

			Weight loss (%)
Entry	Steps	Temp. range (°C)	Exp.	Calc.	m.p. (°C)	Eliminated moiety
Nal	First	203–354	92.44	97.34	232
[Cd2(nal)2(phen)2Cl2]	First	257–457	46.93	46.75	288.9	C26H26CdClN4O3
	Second	457–899	35.29	36.37
	Residue		17.78	16.98		CdO

Fig. 2. TGA pattern of (a) nal and (b) [Cd₂(nal)₂(phen)₂Cl₂] under oxygen atmosphere.

3.5 Single-crystal analysis

The crystal of the complex contains dimeric [Cd(nal) (phen)Cl]₂ molecules having two hexacoordinated Cd(II) metal ions. The crystal system is triclinic with space group P-1. A diagram of [Cd₂(nal)₂(phen)₂Cl₂] with thermal ellipsoids at 50% is shown in Figure 3, where atomic labeling represents equivalent atoms generated from the symmetry code . Selected bond distances and angles are listed in Table 2. The structure was solved by the direct method using SHELXS-86 and refined with SHElXL-97. The nal behaves as a bidentate deprotonated ligand and is coordinated to Cd(II) through pyridone oxygen and a carboxylate oxygen. Each metal environment is formed by two nitrogen atom of phen, one pyridone oxygen atom of one nal, one carboxylate oxygen atom of the same nal, one carboxylate oxygen atom of another nal and one chlorine atom. In the overall crystal structure, two chlorine atoms are trans to each other. In this crystal, carboxylate oxygen atom of each nal forms a bridge between two cadmium metal ions. Three of the Cd˭O distances ranges from 2.3582(15) to 2.3676(17) Å, which is different to those of the reported dinuclear cadmium compound [Cd₂(Cx)₄(H₂O)₂] [36].

Table 2  Bond lengths (Å) and angles (°) of [Cd₂(nal)₂(phen)₂Cl₂] around Cd(II).

N(1)–Cd(01)	2.3521(16)	N(1)–Cd(01)–N(2)	70.61(6)
N(2)–Cd(01)	2.3601(18)	O(2)#1–Cd(01)–N(2)	91.20(5)
O(2)–Cd(01)	2.2919(15)	O(2)–Cd(01)–O(4)#1	143.28(5)
O(2)–Cd(01)#1	2.3582(15)	N(1)–Cd(01)–O(4)#1	83.07(5)
O(4)–Cd(01)#1	2.3676(17)	O(2)#1–Cd(01)–O(4)#1	73.31(5)
Cl(1)–Cd(01)	2.4856(11)	N(2)–Cd(01)–O(4)#1	85.49(5)
Cd(01)–O(2)#1	2.3582(15)	O(2)–Cd(01)–Cl(1)	102.34(4)
O(2)–Cd(01)–N(1)	130.42(5)	N(1)–Cd(01)–Cl(1)	93.19(5)
O(2)–Cd(01)–O(2)#1	70.11(6)	O(2)#1–Cd(01)–Cl(1)	101.81(5)
N(1)–Cd(01)–O(2)#1	151.35(5)	N(2)–Cd(01)–Cl(1)	163.16(4)
O(2)–Cd(01)–N(2)	92.05(5)	O(4)#1–Cd(01)–Cl(1)	88.04(4)

Note: Symmetry transformations used to generate equivalent atoms: .

Fig. 3. Symmetry-related crystal diagram of [Cd₂(nal)₂(phen)₂Cl₂].

3.6 Biological activity

Antifungal activity of the free ligand and synthesized complex was carried out against four fungi, namely PA, SR, RS, RB. The results of the antimicrobial tests are illustrated graphically in Figure 4. Antimicrobial activity has been evaluated on the basis of size of the inhibition zone around the dishes. It has been observed that the antifungal activity of the newly synthesized complex increases with an increase in concentration, but remains the same for the parent ligand. The complex shows more activity as compared with the standard ligand indicating that metal complexation enhances the activity of the parent ligand. This may be explained by the chelation theory [47], which depicts that the delocalization of π electrons over the whole chelate ring increases, due to which polarity of the ligand and the central metal atom decreases which results in the penetration of the complex through the lipid layer of the cell membrane.

Fig. 4. Percentage inhibition of (a) nal and (b) [Cd₂(nal)₂(phen)₂Cl₂] against PA, SR, RS and RB.

4. Conclusions

The synthesis and the characterization of neutral binuclear mixed ligand metal complex of nal and phen with Cd(II) metal ion have been realized with physicochemical and spectroscopic methods. The ligand is bonded to Cd(II) ion via the pyridone oxygen and one carboxylate oxygen. A distorted octahedral environment around Cd(II) has been suggested from the single-crystal XRD data. The antifungal activity of the complex has been tested on four different fungi and the results have shown an enhanced biological activity in comparison with the free ligand.

Funding

The authors are pleased to acknowledge University Grants Commission (UGC), Delhi, India, for financial support.

The authors acknowledge National Centre of Integrative Pest Management, PUSA, India, for the screening of the drugs and Department of Chemistry, University of Delhi for providing necessary facilities.